Embryos of many plant seeds accumulate sucrose and the raffinose family of oligosaccharides (RSO), such as raffinose, stachyose and verbascose, as the major soluble sugars in mature seeds (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994); Obendorf, See Sci. Res. 7:63-74 (1997)). Soybean (Glycine max (L.) Merrill) seeds accumulate soluble carbohydrates, primarily sucrose, raffinose, and stachyose and lesser amounts of galactopinitol A, galactopinitol B, ciceritol, and fagopyritol B1 in axis and cotyledon tissues as part of the seed maturation process (Obendorf et al., Crop Science 38:78-84 (1998)). By contrast, embryos of maturing buckwheat (Fagopyrum esculentum Moench) seeds accumulate fagopyritols, galactosyl derivatives of D-chiro-inositol, instead of raffinose and stachyose (Horbowicz et al., Planta 205:1-11 (1998)). Six fagopyritols, in two different series, are present in buckwheat embryos: fagopyritol A1 (α-D-galactopyranosyl-(1→3)-1D-chiro-inositol), fagopyritol A2 (α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→3)-1D-chiro-inositol), fagopyritol A3(α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→3)-1D-chiro-inositol), fagopyritol B1 (α-D-galactopyranosyl-(1→2)-1D-chiro-inositol), fagopyritol B2 (α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→2)-1D-chiro-inositol), and fagopyritol B3 (α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→2)-1D-chiro-inositol) (Horbowicz et al., Planta 205:1-11 (1998); Szczecinski et al., Bull. Polish Acad. Sci., Chem. 46:9-13 (1998); Obendorf et al., Carbohydr. Res. 328:623-627 (2000); Steadman et al., Carbohydr. Res. 331:19-25 (2001)). Fagopyritols are concentrated in the axis and cotyledon tissues of embryos in mature buckwheat seeds (Horbowicz et al., Planta 205:1-11 (1998)). Buckwheat bran, a commercial milling fraction (Steadman et al., J. Cereal Sci. 33:271-278 (2001)), is a rich source of fagopyritols (Steadman et al., J. Agric. Food Chem. 48:2843-2847 (2000)).
Fagopyritols are of considerable interest for the treatment of non-insulin dependent diabetes mellitus (NIDDM) and polycystic ovary syndrome (PCOS), both insulin response disorders. Fagopyritol A1 is isosteric with 2-amino-2-deoxy-α-D-galactopyranosyl-(1→3)-1D-chiro-inositol (Berlin et al., Tetrahedron Lett. 31:1109-1112 (1990)) related to a putative insulin mediator (Berlin et al., Tetrahedron Lett. 31:1109-1112 (1990); Lamer et al., Biochem. Biophys. Res. Comm. 151:1416-1426 (1988)) deficient in subjects with NIDDM (Fonteles et al., Diabetologia 39:731-734 (1996); Lamer et al., Diabetes Rev. 7:217-231 (1999)) and PCOS (Nestler et al., J. Clin. Endocrin. Metab. 83:2001-2005 (1998); Nestler et al., New England J. Med. 340:1314-1320 (1999); Nestler et al., J. Pediatric Endocrin. Metab. 13(Suppl. 5):1295-1298 (2000)).
Enzymes (fagopyritol synthases) catalyzing the biosynthesis of fagopyritols in buckwheat or other plants have not been described. The present invention is directed to overcoming this and other deficiencies in the prior art. SUMMARY OF THE INVENTION
The present invention relates to isolated nucleic acid molecules which encode a fagopyritol synthase and the amino acid sequences encoded by such nucleic acid molecules.
Another aspect of the present invention pertains to host cells, expression vectors, transgenic plants, and transgenic plant seeds containing the isolated nucleic acid molecules of the present invention.
The present invention is also directed to a method for producing a fagopyritol, an insulin mediator, an insulin mediator analogue, or an insulin mediator homologue. This method includes providing a fagopyritol synthase, providing a substrate including a galactosyl donor and a galactosyl acceptor, and combining the fagopyritol synthase with the substrate under conditions effective to produce a fagopyritol, an insulin mediator, an insulin mediator analogue, or an insulin mediator homologue.
The fagopyritol synthases of the present invention can be used to produce fagopyritols, insulin mediators, insulin mediator analogues, or insulin mediator homologues which can be used in a pharmaceutical composition which also includes a pharmaceutical carrier. This pharmaceutical composition or, alternatively, the fagopyritols, insulin mediators, insulin mediator analogues, or insulin mediator homologues can be administered to a patient to treat disorders, such as diabetes and PCOS. In addition, the fagopyritol synthases can be used to produce transgenic plants useful for nutraceutical applications.